Isolation of novel bacteria contributing to soilborne disease suppression

ABSTRACT

Embodiments relate to plant disease suppressive microorganisms and compositions including the same, methods for isolating disease suppressive microorganisms, and methods for controlling plant diseases using disclosed compositions and microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Patent Application No. 61/083,766, filed Jul. 25, 2008, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

Embodiments relate to compositions comprising plant disease suppressive microorganisms, methods for isolating disease suppressive microorganisms, and methods for controlling plant diseases using disclosed compositions and microorganisms.

BACKGROUND OF THE ART

Soilborne plant pathogenic fungi and oomycetes cause severe economic losses in the agricultural and horticultural industries. For example, root and crown rot diseases caused by pathogens such as different Pythium spp. are a widespread and recurrent problem in plant production. As another example, Rhizoctonia solani is a major soilborne fungal phytopathogen, and is associated with diseases such as damping-off, root rot, and leaf and stem rot in many plant species, including greenhouse crops. R. solani is also associated with brown patch in creeping bentgrass and various other turfgrasses of high commercial value. Species of Alternaria and Fusarium are associated with diseases such as early blight of tomato and Fusarium wilt of numerous fruit and vegetable crops.

In light of actual and potential environmental and health hazards associated with pesticide use, chemical fungicide use may be restricted. And, certified organic growers may not use synthetic chemicals for pest management. As a result, growers have sought alternative approaches to disease control. These alternative approaches include the use of biological agents and disease-suppressive growing media. The use of biologically active agents in the control of plant pests and diseases has become especially important. Despite the recent commercialization of several types of microbial biocontrol agents, questions still remain about the ability of these agents to provide consistent and reliable control against pathogens.

SUMMARY OF THE INVENTION

Embodiments relate to plant disease suppressive microorganisms, and methods for the isolation of the same. Also disclosed are methods of using the disclosed compositions for controlling plant diseases.

An example embodiment provides a biologically pure strain of a plant disease suppressive microorganism. A preferred embodiment comprises a disease suppressive strain designated H24L5A, deposited as ATCC PTA-10183. An alternative embodiment comprises the disease suppressive strain designated R4F2, deposited as ATCC PTA-10182. In another embodiment, ATCC PTA-10183 and ATCC PTA-10182 are used in combination. Additional embodiments comprise an isolated strain harboring a 16S ribosomal RNA gene comprising at least 97% sequence identity to a sequences identified in Table 2.

Exemplary embodiments also include novel compositions for the biological control of plant pathogens. In some embodiments, a composition may comprise an inert carrier and bacteria of a strain that exhibits fungicidal or fungistatic activity. A composition can also include a growth medium. In other embodiments, the composition may comprise a novel bacterium deposited as ATCC Accession No. PTA-10183. In an alternative embodiment, the composition may comprise a bacterium stain, deposited as ATCC Accession No. PTA-10182. In other embodiments, the composition may comprise ATCC Accession No. PTA-10183 and ATCC Accession No. PTA-10182. In additional embodiments, the composition may comprise an isolated bacterial strain harboring a 16S ribosomal RNA gene comprising at least 97% sequence identity to a sequence identified in Table 2. In various embodiments, compositions may also comprise a growth medium and metabolites produced by the strains noted above.

The novel compositions and methods can be used, for example, to suppress diseases associated with soilborne plant pathogenic fungi, e.g., Rhizoctonia species such as R. solani. The novel compositions and methods can also be effective in suppressing diseases associated with various plant pathogenic oomycetes (e.g. Pythium, Phytophthora), fungi (e.g. Alternaria, Colletotrichum and Fusarium), and bacteria (e.g. Pseudomonas, Xanthomonas).

Exemplary embodiments include methods for the identification and isolation of bacteria responsible for plant disease suppression, particularly novel members of the Mitsuaria and Burkholderia species. More specifically, various embodiments utilize sequences and terminal restriction fragments (TRF) of 16S rDNA statistically associated with damping-off disease suppression to identify and isolate bacteria giving rise to those TRF.

Accordingly, embodiments provide a method for the isolation of bacteria contributing to soilborne plant disease suppression comprising:

a) identifying a terminal restriction fragment (TRF) of 16S rDNA statistically associated with a soilborne plant disease suppression activity;

b) cloning the TRF identified in step (a) to obtain a cloned TRF;

c) sequencing the TRF identified in step (a) to obtain a TRF sequence;

d) selecting a TRF primer specific to the cloned TRF;

e) selecting a downstream 16S rDNA primer;

f) screening pools of cultured isolates using the TRF specific primer and the a downstream 16S rDNA primer for the presence of an amplification product;

g) sequencing the 16S rDNA of an amplification-product-positive colony to obtain a colony specific sequence;

h) comparing the colony specific sequence to the TRF sequence; and

i) isolating the amplification-product-positive colony if the sequences in step (h) are essentially identical.

In another aspect, embodiments feature compositions comprising a bacterial strain that exhibits fungicidal or fungistatic activity combined with an inert carrier. The bacterial strain is present at about 10⁵ cfu to about 10¹¹ cfu per gram of carrier. Such a composition can be in the form of a granule, wettable powder, or liquid concentrate. In some embodiments, the bacterial strain(s), e.g., the bacterial strains deposited as ATCC Accession No. PTA-10183, ATCC Accession No. PTA-10182, or a combination thereof, exhibits fungicidal or fungistatic activity towards a fungal plant pathogen. The pathogens against which fungicidal or fungistatic activity is observed can be, for example, a species of Rhizoctonia, Pythium, Phytophthora, Fusarium, Alternataria, or Colletotrichum.

The invention also features a method of controlling or suppressing the growth of a plant pathogenic fungus. In some embodiments, the method comprises applying an effective amount of a bacterial strain designated ATCC Accession No. PTA-10183, ATCC Accession No. PTA-10182, or a combination thereof, to an environment in which the plant pathogenic fungus may grow. Additional embodiments comprise applying an effective amount of a bacterial strain harboring a 16S ribosomal RNA gene comprising at least 97% sequence identity to a sequences identified in Table 2. In other embodiments, the method comprises applying an effective amount of a composition to an environment in which the plant pathogenic fungus may grow. Such a composition comprises a bacterial strain that exhibits fungicidal or fungistatic activity combined with an inert carrier. The composition can include a growth medium and metabolites of the bacterial strains noted above. The fungus may be a species of Rhizoctonia, Pythium, Phytophthora, Fusarium, Alternataria, or Colletotrichum.

Various embodiments also feature a method of controlling the growth of a plant pathogenic fungus. The method involves applying a composition to a plant. The composition comprises a bacterial strain that exhibits fungicidal or fungistatic activity combined with an inert carrier and, optionally, bacterial metabolites and/or a growth medium. The bacterial strain may be ATCC Accession No. PTA-10183, ATCC Accession No. PTA-10182, or a combination thereof. In additional embodiments, the composition may comprise an isolated bacterial strain harboring a 16S ribosomal RNA gene comprising at least 97% sequence identity to a sequence identified in Table 2. In the method, symptoms of a disease caused by the fungus are ameliorated or suppressed on the plant. The composition can be applied to the leaves or stem of the plant, e.g., the leaves or the stem of a vegetable crop.

Embodiments also features a method of controlling the growth of a plant pathogenic fungus, which comprises applying a composition to seed or soil. The composition comprises a bacterial strain that exhibits fungicidal or fungistatic activity combined with an inert carrier and, optionally, bacterial metabolites and/or a growth medium. The bacterial strain can be ATCC Accession No. PTA-10183, ATCC Accession No. PTA-10182, or a combination thereof. In additional embodiments, the composition may comprise an isolated bacterial strain harboring a 16S ribosomal RNA gene comprising at least 97% sequence identity to a sequence identified in Table 2. In the method, symptoms of a disease associated with the fungus are ameliorated or suppressed on a plant growing in the soil. The fungus can be a species of Rhizoctonia, Pythium, Phytophthora, Fusarium, Alternataria, or Colletotrichum.

BRIEF DESCRIPTION OF THE BIOLOGICAL DEPOSITS AND SEQUENCE DESCRIPTIONS

The various embodiments of the invention can be more fully understood from the following detailed description, biological deposits, and the accompanying sequence descriptions, which form a part of this application.

Applicants made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure:

International Depositor Identification Depository Reference Designation Date of Deposit H24L5A PTA-10183 Jul. 8, 2009 R4F2 PTA-10182 Jul. 8, 2009

The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

TABLE 1 Primers and oligonucleotide adapters Description Name Sequence (SEQ ID NO ) Mspl TRF Mspl- 5′-CGGTACTCAGGACTCAT-3′ cloning adapter 1 (SEQ ID NO: 1) Mspl- 5′-GACGATGAGTCCTGAGTAC-3′ adapter 2 (SEQ ID NO: 2) Mspl- 5′-GATGAGTCCTGAGTACCG-3′ adapter (SEQ ID NO: 3) primer Variable M139F 5′-TAACGCGGGGCAACCTGGCGA-3′ loop- (SEQ ID NO: 4) specific M141F 5′-CAGCACGGGAGCAATCCTGGTGG-3′ (SEQ ID NO: 5) M141-F2 5′-GGAGCAATCCTGGTGGCGA-3′ (SEQ ID NO: 6) 16S 8F 5′-AGAGTTTGATCCTGGCTCAG-3′ amplifi- (SEQ ID NO: 7) cation 1492R 5′-ACGGCTACCTTGTTACGACTT-3′ (SEQ ID NO: 8) 518R 5′-ATTACCGCGGCTGCTGG-3′ (SEQ ID NO: 9)

While the primer sequences and adapters identified in Table 1 were used in the examples below, it is appreciated that embodiments include all primers that might reasonably bind to the 16S sequences listed as SEQ ID NOS: 10-25.

SEQ ID NOs: 10-25 are the nucleotide sequences of the 16S rRNA genes of plant disease suppressive strains, isolated as described in the examples below.

TABLE 2 Genbank Accession Corresponding Isolate SEQ ID Number Sequence Description Designation SEQ ID NO 10 EU714905 Mitsuaria sp. 16S ribosomal RNA gene, H24L5A (PTA-10183) partial sequence SEQ ID NO 11 EU714906 Mitsuaria sp. 16S ribosomal RNA gene, H24L3B partial sequence SEQ ID NO 12 EU714907 Mitsuaria sp. 16S ribosomal RNA gene, H23L1 partial sequence SEQ ID NO 13 EU714908 Mitsuaria sp. 16S ribosomal RNA gene, H24L2C2 partial sequence SEQ ID NO 14 EU714909 Mitsuaria sp. 16S ribosomal RNA gene, H24L1B partial sequence SEQ ID NO 15 EU714910 Mitsuaria sp. 16S ribosomal RNA gene, H24L1C partial sequence SEQ ID NO 16 EU714911 Mitsuaria sp. 16S ribosomal RNA gene, H24L6B partial sequence SEQ ID NO 17 EU714912 Mitsuaria sp. 16S ribosomal RNA gene, H29L1B partial sequence SEQ ID NO 18 EU714913 Burkholderia sp. 16S ribosomal RNA R2G3 gene, partial sequence SEQ ID NO 19 EU714914 Burkholderia sp. 16S ribosomal RNA R4F2 (PTA-10182) gene, partial sequence SEQ ID NO 20 EU714915 Burkholderia sp. 16S ribosomal RNA R4G3 gene, partial sequence SEQ ID NO 21 EU714916 Burkholderia sp. 16S ribosomal RNA R4C3 gene, partial sequence SEQ ID NO 22 EU714917 Burkholderia sp. 16S ribosomal RNA R4F3 gene, partial sequence SEQ ID NO 23 EU714918 Burkholderia sp. 16S ribosomal RNA R4A2 gene, partial sequence SEQ ID NO 24 EU714919 Burkholderia sp. 16S ribosomal RNA R4E2 gene, partial sequence SEQ ID NO 25 EU714920 Burkholderia sp. 16S ribosomal RNA R2C2 gene, partial sequence

Other features and advantages will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the embodiments will be obtained from a reading of the following detailed description and the accompanying drawings in which:

FIG. 1 is a DNA-sequence alignment showing the position and variation of the first variable region of the 16S rRNA of representative species within the order Burkholderiales and clones generated in this study. E. coli sequence is shown as a reference, with the variable loop between positions 69 and 101. Primers were designed for this region. Primer sequences and overlap are shown below the alignment.

FIG. 2 is a classification chart of the M139-associated isolates (□) as Mitsuaria sp. based on 16S rDNA sequence analyses. Included in the dendrogram are the sequence of the type strains representative of other species of Genera incertae of the order Burkholderiales. The phylogenetic relationships among taxa were inferred from ˜1200 bp of the 16S rDNA gene, using the Neighbor-Joining method from distances computed by the Maximum Composite Likelihood algorithm. Bootstrap values >60% (1000 replicates) are shown next to the branches. Accession numbers for each sequence are shown in parenthesis. Scale bar: number of base substitutions per site.

FIG. 3 is a classification chart of M141-associated isolates (⋄) as a novel Burkholderia sp. based on 16S rDNA sequence analyses. Included in the dendrogram are the sequence of the other 22 named Burkholderia species. The phylogenetic relationship among taxa was inferred from ˜1300 bp of the 16S rDNA gene, using the Neighbor-Joining method from distances computed by the Maximum Composite Likelihood algorithm. Bootstrap values >60% (1000 replicates) are shown next to the branches. Accession numbers for each sequence are shown in parenthesis. Scale bar: number of base substitutions per site. *Candidatus Burkholderia species with no cultured isolate.

FIG. 4 is a graph of the frequency of positive in-vitro inhibition activity of Mitsuaria (A) and Burkholderia (B) isolates identified in this study against multiple fungal and oomycete tomato and soybean pathogens. In-vitro inhibition activity was tested for eight isolates of each genus on three different media and was scored as positive or negative. TSA, trypticase soy agar; R2A, R2A media for growth of heterotrophic organisms; KB, King's medium B; LM, Leptothrix strain medium.

FIG. 5 shows experimental photographs demonstrating the chitinolytic activity of Mitsuaria isolates.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

A sequence-directed culturing strategy was developed using TRF-derived markers and media reported to be selective for the genera identified. Using exemplary methods, novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted TRF were isolated and purified. The isolated species inhibit growth of multiple plant pathogens, and usually suppress soybean and tomato seedling diseases. Two embodiments, which are believed to include Mitsuaria and Burkholderia stains, were deposited as ATCC Accession No. PTA-10183, ATCC Accession No. PTA-10182, respectively. Both strains displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro, and reducing disease severity of infected tomato and soybean seedlings.

Embodiments include isolated and purified bacterial strains involved in plant pathogen suppression. Specific embodiments include bacterial strains deposited as ATCC Accession No. PTA-10183 and ATCC Accession No. PTA-10182. Embodiments of the invention also include other strains identified in Table 2. Furthermore, embodiments include other strains harboring a 16S ribosomal RNA gene comprising at least 97% sequence identity to the strains identified in Table 2. For example, at least one embodiment includes a biologically pure culture of a bacterial strain comprising a nucleic acid, the nucleic acid comprising a 16S ribosomal RNA gene sequence at least 97% identical to SEQ ID NO: 10, the bacterial strain exhibits plant pathogen suppression when applied to plant material or a soil environment. Furthermore, at least one embodiment includes a biologically pure culture of a bacterial strain comprising a nucleic acid, the nucleic acid comprising a 16S ribosomal RNA gene sequence at least 97% identical to SEQ ID NO: 19, the bacterial strain exhibits plant pathogen suppression when applied to plant material or a soil environment. Rather, specific embodiments encompasses bacteria containing nucleic acid molecules carrying modifications such as substitutions, small deletions, insertions, or inversions, which nevertheless are at least 97% identical (e.g., at least 98% or 99% identical) to the nucleotide sequence shown as SEQ ID NOs: 10 and 19 in the Sequence Listing.

The determination of percent identity or homology between two sequences is accomplished using the algorithm of Karlin and Altschul (1990) Proc. Nat'l Acad. Sci. USA 87: 2264-2268, modified as in Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. For the purposes of this disclosure, determinations of percent identity are computed using the default parameters of BLASTN optimized for highly similar sequences (i.e. megablast) of the respective programs (e.g., XBLAST and NBLAST). See http://www.ncbi.nlm.nih.gov.

Furthermore, various embodiments include compositions and methods for utilizing the identified strains.

Embodiments also include methods for identifying and isolating bacterial strains involved in plant pathogen suppression. Various methods utilize T-RFLP, in conjunction with various other molecular techniques described below, to direct the recovery of novel disease suppressive microbes.

In various embodiments, ATCC Accession No. PTA-10183 and/or ATCC Accession No. PTA-10182 can be used as a solid. For example, a culture of ATCC Accession No. PTA-10183 and/or ATCC Accession No. PTA-10182 is grown in a suitable growth medium, the bacteria separated from the spent medium, resuspended in a fresh medium and the bacteria spray-dried. The resulting powder can be used, e.g., as a dusting biocontrol agent on vegetable crops. Alternatively, ATCC Accession No. PTA-10183 and/or ATCC Accession No. PTA-10182 can be used as a liquid, e.g., a culture of ATCC Accession No. PTA-10183 and/or ATCC Accession No. PTA-10182 can be grown in a suitable growth medium, the bacteria separated from the spent medium, and resuspended in water, buffer or fresh medium. The resulting suspension can be used, for example, as a foliar spray.

In other embodiments, ATCC Accession No. PTA-10183 and/or ATCC Accession No. PTA-10182 can be combined with one or more compounds to form a mixture suitable for applying to an environment in which a plant pathogenic fungus can grow. Compounds that can be combined with ATCC Accession No. PTA-10183 and/or ATCC Accession No. PTA-10182 bacteria include fertilizers, micronutrient donors, surfactants, or adjuvants conventionally employed in the art of formulation. See, e.g., U.S. Pat. Nos. 6,280,719; 5,780,023; 5,765,087; 5,348,742; and 5,068,105. The number of compounds selected for a given mixture may be chosen in accordance with the intended application and/or existing conditions.

The resulting mixture can be a solid or a liquid, e.g., an emulsifiable concentrate, a coatable paste, a directly sprayable solution, a dilutable solution, a dilute emulsion, a wettable powder, a dusting powder, a granular formulation, or an encapsulated formulation.

ATCC Accession No. PTA-10183 and/or ATCC Accession No. PTA-10182 are effective biological control organisms that have fungicidal activity, and may also have fungistatic activity. The isolated embodiments provide good fungal disease suppression. The use of ATCC Accession No. PTA-10183 and/or ATCC Accession No. PTA-10182 as a biocontrol agent may reduce or eliminate the use of environmentally harmful chemical fungicides, especially those derived from petroleum precursors.

Compositions

In various embodiments, bacteria can be combined with an inert carrier to form a composition suitable for applying to soil. For example, compositions comprising ATCC Accession No. PTA-10183 and/or ATCC Accession No. PTA-10182 can be made in accordance with those described in U.S. Pat. No. 6,995,007, incorporated by reference in its entirety.

Bacteria for use in a composition of the invention exhibit fungicidal or fungistatic activity against one or more fungal pathogens of plants. For example, bacteria exhibiting fungicidal or fungistatic activity against a fungal plant pathogen can be used to inhibit growth of that pathogen and thus provide effective biological control.

It is contemplated that a proportion of the bacteria in exemplary compositions can be relatively innocuous bacterial strains that do not exhibit significant fungicidal or fungistatic activity. Relatively innocuous bacterial strains may be advantageous in some embodiments, e.g., as a marker for persistence in the environment or as a marker for effective coverage following spray application of a composition.

In some embodiments, a growth medium is also included in the composition, e.g., a composition of the invention includes bacteria, porous ceramic particles and a growth medium. Without being bound by theory, it is believed that a composition that includes a growth medium provides the bacterium with a nutrient-rich micro-environment, resulting in a competitive advantage to bacteria present in the composition compared to native soil bacteria thus enabling bacteria of the composition to function more effectively as biocontrol agents.

In some embodiments, an amount of water is present in the composition. For liquid concentrates, water is up to 99% by weight.

Methods of Suppressing Fungal Disease

The invention also features a method comprising applying a composition of the invention to an environment in which a plant pathogenic fungus may grow. Such an environment can be soil, a plant seed, a plant, or a plant part (e.g., leaves, roots, branches and stems). The composition typically is applied in an amount effective to control or suppress fungal growth, e.g., in an amount sufficient to control or suppress observable symptoms on a plant of a fungal disease. The rate of application may vary according to the plant species to be protected, the efficacy of the bacterial strain against the pathogen to be controlled, and the severity of the disease pressure. Typically, the rate of application is about 1.3×10³ cfu/cm² to about 1.3×10⁸ cfu/cm² of soil or plant surface area, or about or about 1.3×10³ cfu to about 1.3×10⁸ cfu per seed or cutting. Like the nature of the composition, a method of application such as spraying, atomizing, dusting, scattering or pouring, is chosen in accordance with the intended objectives and the prevailing circumstances.

Particularly suitable methods for applying a composition include methods that involve seed coating, soil application or incorporation into a growth medium. The number of times that a composition is applied may vary, depending on the observed or expected intensity of infestation by a particular fungal pathogen. A composition can be applied to soil as a liquid, but can also be applied to soil in granular form. Outdoor soil applications can be in furrow, broadcast, or soil injection. In greenhouse or other indoor environments, a composition can be applied by mixing with potting soils typically used in such environments. A composition may also be applied to seeds by impregnating the seeds with a liquid formulation, or coating them with a solid formulation. In various embodiments, liquid suspensions of bacteria (in water or a growth media) may be applied to seed at a rate of 5 to 10 ml per kg of seed and allowed to dry prior to bagging and storage. In special cases, further types of application are also possible, for example, selective treatment of individual plant stems or buds.

A suitable group of plants with which to practice the invention include dicots, such as safflower, alfalfa, soybean, or sunflower. Also suitable are monocots such as corn, wheat, rye, barley, or oat. Also suitable are vegetable crops or root crops such as potato, broccoli, peas, peppers, lettuce, sweet corn, popcorn, tomato, beans (including kidney beans, lima beans, dry beans, green beans) and the like. Thus, the invention has use over a broad range of plants, including species from the genera Agroslis, Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypiuni, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panicum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, Poa, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna and Zea.

Plant pathogenic fungi whose disease symptoms can be controlled or suppressed include Pythium aphanidermatum, Phytophthora capsicum, Rhizoctonia solani, Fusarium graminearum, Fusarium oxysporum, and Alternaria solani. Diseases associated with these fungi include damping-off and root rots of multiple plant species. The broad spectrum activity reported here further indicates the utility of the strains against most fungal and oomycete plant diseases.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

General Methods

Standard recombinant DNA and molecular cloning techniques used in the examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987.

Materials and methods suitable for the maintenance and growth of bacterial cultures are also well known in the art. Techniques suitable for use in the following Examples may be found Atlas, R M (1997) Handbook of Microbiological Media, ed Lawrence C. Parks (CRC Press Inc., United States of America), pp 1706; Manual of Methods for General Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Washington, D.C., 1994; or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass., 1989. Example Media include:

Leptothrix strain medium—(LM) per liter: 5 g peptone, 0.2 g magnesium sulfate heptahydrate, 0.15 g ferric ammonium citrate, 0.05 g calcium chloride, 0.01 g ferric chloride anhydrous, 0.01 g manganese sulfate monohydrate, 15 g agar. Yeast agar van Niel's—(YAN) per liter: 10 g yeast extract, 1 g dipotassium phosphate, 0.5 g magnesium sulfate heptahydrate, 15 g agar. Nutrient agar buffered—(NB) per liter: 4 g peptone, 4 g sodium chloride, 2 g yeast extract, 1 g beef extract, 0.45 g monopotassium phosphate, 1.78 g disodium hydrogen phosphate heptahydrate, 15 g agar. King's Medium B—(KB) per liter: 20 g proteose peptone, 1.5 g dipotassium phosphate, 1.5 g magnesium sulfate heptahydrate, 10 ml glycerol, 15 g agar.

Cloning of MspI Generated 16s rDNA TRF

The procedure for cloning and sequencing of TRF was modified from Widmer et al (47). The 16S rDNA was amplified and digested with MspI (Promega) from multiple soil and rhizosphere DNA samples of tomato and soybean (from 14). A double stranded asymmetric adapter was ligated into the MspI site of the TRF. 5 μM MspI-adapters 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) (Table 1) into 1× Buffer C (Promega) and incubating 10 min at 65° C., 10 min at 37° C., 10 min at 25° C. and 10 min at 4° C. For ligation, 2 μl of the digested amplicon were mixed with 1 μl double-stranded adapter, 4.5 U T4 ligase (Promega) and 1× ligase buffer (Promega) in a 10 μl reaction. The reaction was incubated 12 h at 16° C. Following ligation, TRF were size selected from a portion of the agarose gel corresponding to 90 to 160 bp in length and purified using UltraClean GelSpin DNA Purification Kit (MoBio). The purified DNA was used to enrich the samples with 16S rDNA TRF of the target sizes. PCR was performed using 16S primer 8F (SEQ ID NO: 7) in combination with MspI-adapter primer (SEQ ID NO: 3). Amplification was carried out in 25 μl reactions containing 1× Mg-free buffer, 1.8 mM MgCl₂, 0.2 mM dNTPs, 1 pmol μl⁻¹ each primer, 0.04 mg ml⁻¹ RNase A (Novagen), 0.06 U μl⁻¹ Go Taq Flexi DNA polymerase (Promega), and 2.5 μl template. The cycling program consisted of a 5 min at 95° C. followed by 26 cycles of 94° C. for 45 s, 54° C. for 45 s, and 70° C. for 45 s; and an 8 min final extension at 70° C. The double stranded adapter was removed by digestion with MspI and the TRF-enriched samples were ligated into pGEM-T Easy Vector (Promega) prior to introduction into E. coli JM109 competent cells (Promega). A total of 56 transformants were selected for sequencing, based on insert size. Sequencing of this and other samples were performed at the Molecular and Cellular Imaging Center of the OARDC (Wooster, Ohio) in an ABI Prism 3100xl genetic analyzer system using 3′-BigDye dideoxynucleotide triphosphates labeling chemistry.

Extension of Target 16s rDNA TRF

The cloned TRF sequences overlap with the first variable loop region between E. coli positions 69-101 bp (15, www.rna.icmb.utexas.edu). Sequence alignments were used for designing variable loop-specific primers M139F (5′-TAACGCGGGGCAACCTGGCGA-3′) (SEQ ID NO: 4) and M141F (5′-CAGCACGGGAGCAATCCTGGTGG-3′) (SEQ ID NO: 5) (FIG. 1). These primers were used independently in combination with universal primer 518R (SEQ ID NO: 9) to generate extended amplicons from multiple samples, with the following variations in the cycling program: 30 cycles of 94° C. for 1 min, 65° C. for 45 s, and 70° C. for 45 s. Amplicons from two independent samples were cloned, as described above, and 16 transformants were selected for sequencing.

Culture-Based Screening for M139 abd M141 Positive Isolates

A bacterial collection was generated from the rhizosphere of hay grown in soils previously described as suppressive (13, 14). The hay mix contained Festulolium duo (36% v/v), alfalfa (14%), Starfire red clover (11%), Jumbo white clover (9%), Tekapo orchard grass (9%), Tuukka timothy (9%), Lancelot plantain (6%) and chicory (6%). The hay was grown in the greenhouse with during the spring of 2007, with temperatures for the period ranging from 23° C. and 31° C. Roots and soils were thoroughly mixed, and five grams of the mixture was sampled and diluted in 50 ml of sterile water (SW). The suspension was vortexed (1 min), sonicated (1 min), and vortexed again (15 sec), and serially diluted in SW and spread-plated in Leptothrix strain medium (LM), Yeast agar van Niel's (YAN), Nutrient agar buffered (NB) and R2A (Difco BD). These culture media were previously reported to support the growth of various Burkholderiales species, including members of the Commamonadaceae (R2A and NB) and Genera Incertae Sedis (R2A, LM, YAN). Plates were incubated for 48 h at room temperature (RT) in the dark. From each plate eight colonies were picked and transferred into a 96-well plate pre-filled with 200 μl well⁻¹ of corresponding liquid medium. A total of 11 mixed hay pots were sampled, resulting in a collection of 704 isolates. Liquid cultures were pooled (eight per well), prior to DNA isolation performed with the Wizard Genomic DNA purification kit (Promega). DNA-pools (1:100 dilution) were PCR-screened for the presence of M139 and M141-like sequences as described above, with a 25 cycles amplification program. The primer and amplification protocol for M141 was modified (M141F2-primer: 5′-GGAGCAATCCTGGTGGCGA-3′ (SEQ ID NO: 6); amplification reaction with final 1.0 mM MgCl₂) to maximize recovery of isolates matching the targeted variable loop sequence. Individual amplifications were performed from individual cultures present in PCR-positive pools only. Colony-PCR was performed with the 8F and 1492R primer combination (14). 16S amplicons were purified with ExoSAP-IT (USB), and sequenced. Consensus sequences for each isolate were constructed using Sequencher 4.7 (Gene Codes Corporation).

In vitro Inhibition of Pathogen Growth

Pathogen growth inhibition was tested in multiple contexts. For Mitsuaria isolates, assays were performed on R2A, LM and 1/10 TS agar (TSA). For Burkholderia isolates, R2A, LM and 1/3 King's Medium B (KB, (54)) were used. Bacteria from 48 h-old culture plates were resuspended in SW, and a 10 μl drop was placed on a plate with a test pathogen in the center. Plates were incubated at RT and growth inhibition was scored between 4-10 days, depending on the pathogen. In vitro inhibition was scored as positive or negative, though phenotypes scored as positive varied somewhat depending on the pathogen and media combination used. Positive scores reflected the formation of clear inhibition zones between the pathogen and the bacteria, diminished total growth of pathogen as compared to the control, melanization or morphology change in pathogen colony, and/or bacterial swarming over the pathogen culture. In vitro inhibition tests were performed against Pythium aphanidermatum isolate 349, and Phythopthora capscici provided by S. Miller (OARDC); Pythium sylvaticum 134, Phythophthora sojae race 25 and Rhizoctonia solani AG4 provided by A. Dorrance (OARDC); F. graminearum provided by P. Paul (OARDC); and Alternaria solani Mg23 and Fusarium oxysporum Ft25 (59).

Seedling Disease Bioassays

Soybean and tomato seeds were surface sterilized and germinated on water agar (WA; 7.5 g agar I⁻¹) at RT in the dark. After four days three seedlings were transferred to Petri-plates containing WA (tomato: 100×15 mm; soybean 150×15 mm). A 5 mm pathogen plug was placed in the center of the plate and seedlings were treated with ˜10⁷ cell ml⁻¹ seedling⁻¹, in ≦100 ul volume. Inoculum was prepared from 24 h cultures in 1/10×TS broth, collected by centrifugation, and washed twice with SW. Control plates with water-treated seedlings with and without pathogen inoculum were also prepared. Each plate was prepared in triplicate. Seedling disease was scored after 4 and 5 days for soybean and tomato respectively. For each (n≧9) seedling, total seedling length and lesion length were measured, and disease severity was expressed as the percent of the seedling that showed a lesion. Three bacterial isolates of each recovered genus were selected for analysis based on their independent isolation from different hay-containing pots. For Mitsuaria isolates, soybean assays were run against P. aphanidermatum, P. sojae and R. solani and for tomato against P. aphanidermatum and R. solani. For Burkholderia isolates soybean and tomato assays were run against R. solani only. All experiments were run at least twice.

Sequence Analyses

Sequences were aligned and pair-wise comparisons calculated with ClustalW2 (EMBL-EBI Tools). Graphic alignments were prepared using Jalview (v 2.3) alignment editor. Individual sequences were compared to the non-redundant nucleotide collection NCBI database (nr/nt, as of Mar. 8, 2008) using blastn. Phylogenetic analyses were performed using MEGA 4. Tree topologies generated by different algorithms were compared and found to be equivalent (data not shown). Sequences were deposited in GenBank with Accession No. EU714905-EU714956.

Statistical Analyses

All analyses were performed using JMP v7.0 (SAS Institute Inc.). The Kruskall-Wallis test was used to determine differences in disease severity. Five treatment levels were considered: three bacterial isolates in the presence of pathogen and water treated seedlings with or without pathogen. Pair-wise comparisons were performed between individual bacteria and water treated seedlings (plus pathogen) with Wilcoxon-2-sample test. Contrast analysis (Wilcoxon-2-sample test, one tail) was performed to determine overall effect of bacterial treatment compared to water treated seedlings (plus pathogen).

Example 1 Classification of 16S Eubacterial Sequences Corresponding in Size to a Target TRF

The identity of bacteria giving rise to MspI generated TRF associated with disease suppression in the microbial community profiles (see Benítez M, et al (2007) Multiple statistical approaches of community fingerprint data reveal bacterial populations associated with general disease suppression arising from the application of different organic field management strategies. Soil Biology and Biochemistry 39, 2289-2301, incorporated by reference in its entirety) was first assessed by cloning TRF of the selected size range. Of 56 clones sequenced, 20 were confirmed as a targeted TRF (seven to M139, eight to M141 and five to M148). These sequences were compared to GenBank using blastn. Five M139 clones shared >90% sequence identity with one another, and likely arise from β-Proteobacteria; and, of these, four, recovered from three independent samples, shared >97% sequence identity to database members of the order Burkholderiales not assigned to a named family (i.e. Genera Incertae Sedis). Similarly, four M141 clones derived from independent samples showed a high degree of similarity to one another and were classified as Burkholderiales, but of more diverse origin. Other M141 clones differed substantially from this group (68-82% sequence identity) and among themselves (66-78% sequence identity) and might belong to the divisions Gemmatimonadetes, Acidobacteria and/or Spirochaete. The greatest sequence variation was observed within the sampled population of M148 clones, which shared only 46-71% sequence identity with each other. Two matched Proteobacteria, one matched Spirochaetae and one matched Planctomycete sequences. Within each cloned TRF subset, at least one did not show any significant similarity to any taxonomic group within GenBank. These data further support our initial hypothesis that multiple novel bacterial populations are associated with the suppressive activity developing from the hay-based transition strategy.

The cloned M139 and M141 TRF were used to recover longer and more phylogenetically informative sequences from the suppressive soils. Among these, over half of the TRF likely arose from novel bacterial species not previously associated with plant disease suppression (i.e. Burkholderiales, Genera Incertae Sedis). Sequence alignments of known Burkholderiales species and M139 and M141 clones revealed sequence variation within the first variable loop of the 16S rRNA (15), and this data was used to design M139- and M141-specific primers (FIG. 1; Table 1). These primers were used in combination with eubacterial primer 518R (SEQ ID NO: 9) to generate extended amplicons from two DNA samples from 14. Four of the M139-extended sequences showed similarity to bacteria of the Genera Incertae Sedis (four genera with >97% identity) and three M141 matched Comamonadaceae (2 genera with >97% identity).

Sequences from both cloning steps were aligned to assemble consensus sequences. For M139, three different consensus sequences with 100% identity over a 76 nt overlap were constructed. Based on approximately 520 nt, the three M139 constructed sequences exhibited >97% identity to database entries of Genera Incertae Sedis Leptothrix, Ideonella and Methylibium, respectively. In addition, one M141 consensus sequence was constructed (97% sequence identity on a 78 nt overlap) which exhibited <97% sequence identity to database entries of the Comamonadaceae. Sequence analysis revealed the presence of a MspI recognition site that will produce a TRF of 139 bp in the three Genera Incertae Sedis-like assembled sequences. The Comamonadaceae-like sequence, however, lacked the MspI site to produce the expected 141 bp TRF. It is unclear if this lack of consistency reflects a high degree of sequence diversity amongst the bacteria giving rise to the targeted TRF in our samples or amplification artifacts.

Example 2 Culture-Collection Screening for M139 and M141 Isolates

Because no isolates with 100% sequence identity to the cloned markers had been previously identified, efforts were made to recover bacteria giving rise to the M139 and M141 markers. To do so, culture media favoring growth of Burkholderiales species related to the genera described above were selected. The isolates were obtained from the mixture of hay species that had resulted in damping-off suppression, and a 2-step PCR-based approach was used to screen the collection, first from pooled samples and then individually. Of the 704 isolates examined, eight, all isolated from Leptothrix strain medium had an exact sequence match to the M139 variable loop. The highest BLAST hit to a named species for all eight isolates was to Mitsuaria chitosanitabida (98-99% identity), followed by Roseateles depolymerans and Pelomonas aquatica or P. saccarophila (>97% identity), all belonging to the Genera Incertae Sedis. Sequence identity within the isolates ranged from 98-100%, and their phylogenetic relationships to representative type strains of Genera Incertae Sedis (Burkholderiales) are shown in FIG. 2. The type strain most closely related to the isolates retrieved from the mixed species hay soils is M. chitosanitabida 3001 (17), but there is a clear distinction between known Mitsuaria species and the isolates from this study.

While the novel Mitsuaria isolates recovered from the disease-suppressive soil were found to have 16S sequences that similar to the initial M139 clones, they were not identical. The isolates shared just 99% identity to a Mitsuaria-like extended sequence clones. Mitsuaria species, also, do not produce an M139 in vitro or in silico. In contrast, the MspI TRF for the isolates was 487 nt (488 nt expected from sequence). Interestingly, M488 and M489 TRF were common in the TRF profiles of the studied soils, and positive associations between M488 and M489 and soilborne disease suppression were observed in two of the studied contexts (14). Variation in TRF size could relate to amplification artifacts resulting from sampling complex mixtures of closely related bacteria, as well as to the presence of pseudo-terminal restriction fragments in the samples (18). Given the sequence similarity between Mitsuaria isolates and M139 clones it seems likely that these represent bacteria very closely related to those giving rise to the M139 TRF associated with disease suppression.

A similar isolation strategy led to the recovery of eight pure cultures from R2A media with an M141-like amplification profile. The 16S sequences amplified from these isolates shared 24 of the 26 nt of the M141-derived variable loop sequence. The highest BLAST hit for all eight was to unclassified Burkholderia spp. (i.e. 99% identity to GenBank AY238505, AB025790, and AB298718). Sequence identity within the eight isolates was >99%, but was only 96% identical to the type strain of the genus, B. cepacia (GenBank U96927). The isolates from this study form a phylogenetically-distinct cluster within the genus (FIG. 3), with their closest relatives being Candidatus Burkholderia spp., non-cultured endosymbionts from leaf galls (19, 20; 97% identical). Sequence analysis revealed 97% identity between our Burkholderia isolates and the initial M141 clones, but only 72-88% sequence identity with clones of the ˜450 nt extended sequences. Still, the observed 16S rDNA MspI TRF for the isolates was a 139/141 bp double-peak, indicating that at least one group of bacteria with an M141 TRF was successfully isolated.

Example 3 Characterization of Pathogen Inhibition and Disease Suppressive Activities

The association of the M139 and M141 TRF with in situ soilborne disease suppression (14) led us to hypothesize that the novel Mitsuaria and Burkholderia isolates obtained would express antagonistic activities towards diverse soilborne pathogens. Initially, the capacity of the isolates to reduce pathogen growth in vitro against was assayed. For the Mitsuaria isolates, inhibition was observed regardless of the pathogen tested (FIG. 4A), with the greatest frequency of inhibition expressed against Pythium aphanidermatum Phytophthora sojae, Rhizoctonia solani, and Alternaria solani, and the least against Pythium sylvaticum.

All of the Mitsuaria isolates from this study have chitinolytic activity in vitro (FIG. 5), which can relate to the broad-spectrum inhibition observed against the various fungi. Briefly, for each isolate tested (1-8) 7 μl of bacterial suspension (in water) were spotted on 1/10 TS agar plates amended with 0.2% colloidal chitin. Plates were incubated at room temperature in the dark and observations were recorded at A) 2, B) 5, C) 7 and D) 9 days after inoculation. Pseudomonas fluorescens (Ps., straind wood1R) was used as a negative control for chitinolytic activity. 1: H24LB; 2: H23L1; 3: H24L1C; 4: H24L2C2; 5: H29L1B; 6: H24L5A; 7: H24L6B; 8: H24L3B. Protocol for preparation of colloidal chitin was modified from Rodriguez-Kabana et al. (1983; Plant Soil 75, 95-106) and Shimahara and Takiguchi (1988; Meth Enzymol 161, 417). Briefly, 20 ml of 10N HCl were added to 0.5 g of chitin (Sigma C8908) and stirred constantly for 2 h. The colloidal chitin was thoroughly washed with water, with three over night steps. When suspension reached to pH 6.0 colloidal chitin was resuspended in 200 ml water and stored at 10° C. until use.

Even so, other mechanisms must be involved in the inhibition of the oomycetes which do not harbor chitin as a major component in their cell walls (21). Similar assays were performed with other Mitsuaria spp. including multiple chitosan-degrading strains isolated from soils in Japan (ATCC type strain M. chitosanitabida 3001, strain 12 and strain 13, 17) and gallic acid degrading strains associated to freshwater plants (Mitsuaria spp.: FBTS 25 and FBTS 19, 22). Of these, chitosan-degrading strains 12 and 13 showed a similar spectrum of inhibition; whereas the type strain 3001 gave a positive inhibition in only about half of the assays. While the sequence identity with the tested Japanese strains was ≧98%, the antagonistic phenotype of our isolates was less variable. The Mitsuaria strains recovered from freshwater plants expressed no pathogen inhibition in most cases. Among the Burkholderia isolates, in vitro pathogen inhibition was less frequent and more variable (FIG. 4B). Significant variation in the expressed inhibitory capacities was observed among isolates, with six isolates inhibiting at least three pathogens, but none of these inhibited the same three pathogens. In contrast to Mitsuaria isolates, all eight Burkholderia isolates tested negative for chitinolytic activity.

Seedling diseases were suppressed by inoculation with the novel Mitsuaria isolates. All the tested isolates reduced disease severity in soybeans challenged with P. aphanidermatum (P=0.03 and 0.005) and in tomato challenged with P. aphanidermatum (P=0.0003 and 0.002) and R. solani (P=0.27, 0.02 and 0.0007). Although not significant for most experiments, the lesion severity caused by R. solani was also reduced by the Mitsuaria isolates in three separate assays. Overall, disease severity reductions ranged from 5 to 20 percent (see e.g., Table 2).

TABLE 2 Lesion severity in soybean and tomato seedlings treated with Mitsuaria isolates and challenged with damping-off pathogens Lesion severity^(a) Crop Treatment P. aphanidermatum R. solani Soybean H23L1 44.2^(b)**^(d) 22.9 H24L5A 62.5* 26.7 H29L1B 64.3 33.3 Pathogen 91 32.2 only No Pathogen 27 11.7 K-W test^(c) P < 0.0001 P = 0.0002 Tomato H23L1 57.8** 45** H24L5A 41.7*** 33.3*** H29L1B 60.4** 33.3*** Pathogen 91.7 55.6 only No Pathogen 47.7 34.8 K-W test P = 0.019  P = 0.006  ^(a)Lesion severity, percent of lesion length in relation to seedling length ^(b)Median values are reported, for n = 16 (soybean/R. solani) or n = 12 (others) ^(c)Non-parametric Kruskall-Wallis test was used to assess differences among all five treatments ^(d)Significant pairwise comparisons between treatment and pathogen only control at ***P < 0.01, **P < 0.05 and *P < 0.1 (Wilcoxon 2-sample test).

Though the data represented in Table 2 represent one assay, comparable patterns were observed across experiments. In 7 out of the 11 tests, treatment with Mitsuaria isolate H24L5A resulted in lower disease severity than the water treated control, whereas isolates H23L1 and H29L1B resulted in disease severity reduction in 4 out of the 11 tests, with greatest variation observed in the soybean bioassays (data not shown).

Similarly, seedling disease severity, caused by R. solani was reduced on tomato and soybeans inoculated with Burkholderia isolates. As a group, disease severity was reduced by at least 15% on soybean (P=0.0001 and 0.0005) and 20% on tomato seedlings (P<0.0001 for both tests) compared to the water treated control (Table 3).

TABLE 3 Lesion severity in soybean and tomato seedlings treated with Burkholderia strains and challenged with Rhizoctonia solani Crop Treatment Lesion severity^(a) Soybean R2C2 36.1^(b)***^(d) R2G3 34.5*** R4F2 34.2** Pathogen 56.1 only No pathogen 29.1 K-W test^(c) P = 0.003 Tomato R2C2 46.2*** R2G3 42.3*** R4F2 48.3*** Pathogen 63.6 only No pathogen 45.2 K-W test P = 0.002 ^(a)Lesion severity, percent of lesion length in relation to seedling length ^(b)Median values are reported for n = 12 ^(c)Non-parametric Kruskall-Wallis test was used to assess differences among all five treatments ^(d)Significant pairwise comparisons between treatment and pathogen only control at ***P < 0.01 and **P < 0.05 (Wilcoxon 2-sample test).

For the Burkholderia isolates tested, no apparent variation in their ability to reduce lesion severity was observed. Overall these data support the hypothesis that multiple isolates of novel Mitsuaria and Burkholderia species contribute to the general soilborne disease suppression induced by the mixed hay cropping system.

The following documents are hereby incorporated by reference (there is no admission thereby made with respect to whether any of the documents constitute prior art with respect to any of the claims):

-   1. Torsvik V & Ovreas L (2002) Microbial diversity and function in     soil: From genes to ecosystems. Curr Opin Microbiol 5, 240-245. -   2. Daniel R (2005) The metagenomics of soil. Nature Reviews     Microbiology 3, 470-478. -   3. Van Lanen S G & Shen B (2006) Microbial genomics for the     improvement of natural product discovery. Curr Opin Microbiol 9,     252-260. -   4. Maron P A, Ranjard L, Mougel C & Lemanceau P (2007)     Metaproteomics: A new approach for studying functional microbial     ecology. Microb Ecol 53, 486-493. -   5. Martin H G, et al (2006) Metagenomic analysis of two enhanced     biological phosphorus removal (EBPR) sludge communities. Nat     Biotechnol 24, 1263-1269. -   6. Miller S R, et al (2005) Discovery of a free-living chlorophyll     d-producing cyanobacterium with a hybrid     proteobacterial/cyanobacterial small-subunit rRNA gene. Proceedings     of the National Academy of Sciences 102, 850-855. -   7. Adesina M F, Lembke A, Costa R, Speksnijder A & Smalla K (2007)     Screening of bacterial isolates from various european soils for in     vitro antagonistic activity towards Rhizoctonia solani and Fusarium     oxysporum: Site-dependent composition and diversity revealed. Soil     Biology and Biochemistry 39, 2818-2828. -   8. Mazzola M (2004) Assessment and management of soil microbial     community structure for disease suppression. Annu Rev Phytopathol     42, 35-59. -   9. Borneman J & Becker J O (2007) Identifying microorganisms     involved in specific pathogen suppression in soil. Annu Rev     Phytopathol 45, 153-172. -   10. Baker K F (1987) Evolving concepts of biological control of     plant pathogens. Annu Rev Phytopathol 25, 67-85. -   11. Weller D M, Raaijmakers J M, McSpadden Gardener B B & Thomashow     L S (2002) Microbial populations responsible for specific soil     suppressiveness to plant pathogens. Annu Rev Phytopathol 40,     309-348. -   12. Garbeva P, Postma J, van Veen J A & van Elsas J D (2006) Effect     of above-ground plant species on soil microbial community structure     and its impact on suppression of Rhizoctonia solani AG3. Environ     Microbiol 8, 233-246. -   13. Baysal F, Benitez M, Kleinhenz M D, Miller S A & McSpadden     Gardener B B (2008) Field management effects on damping-off and     early season vigor of crops in a transitional organic cropping     system. Phytopathology 98, 562-570. -   14. Benítez M, et al (2007) Multiple statistical approaches of     community fingerprint data reveal bacterial populations associated     with general disease suppression arising from the application of     different organic field management strategies. Soil Biology and     Biochemistry 39, 2289-2301. -   15. Cannone J J, et al (2002) The comparative RNA web (CRW) site: An     online database of comparative sequence and structure information     for ribosomal, intron, and other RNAs. BMC Bioinformatics 3, 2. -   16. Muyzer G, de Waal E C & Uitterlinden A G (1993) Profiling of     complex microbial populations by denaturing gradient gel     electrophoresis analysis of polymerase chain reaction-amplified     genes coding for 16S rRNA. Appl Environ Microbiol 59, 695-700. -   17. Amakata D, et al (2005) Mitsuaria chitosanitabida gen. nov., sp     nov., an aerobic, chitosanase-producing member of the     ‘Betaproteobacteria’. Int J Syst Evol Microbiol 55, 1927-1932. -   18. Liu, W & Stahl, D A (2007) in Manual of Environmental     Microbiology, eds. Hurst C J, et al (ASM Press) pp 139-156. -   19. Van Oevelen S, De Wachter R, Vandamme P, Robbrecht E & Prinsen     E (2002) Identification of the bacterial endosymbionts in leaf galls     of Psychotria (Rubiaceae, Angiosperms) and proposal of ‘Candidatus     Burkholderia kirkii’ sp nov. Int J Syst Evol Microbiol 52,     2023-2027. -   20. Van Oevelen S, De Wachter R, Vandamme P, Robbrecht E & Prinsen     E (2004) ‘Candidatus Burkholderia calva’ and ‘Candidatus     Burkholderia nigropunctata’ as leaf gall endosymbionts of african     psychotria. Int J Syst Evol Microbiol 54, 2237-2239. -   21. Bartnicki-Garcia S (1968) Cell wall chemistry, morphogenesis,     and taxonomy of fungi. Annu Rev Microbiol 22, 87. -   22. Muller N, Hempel M, Philipp B & Gross E M (2007) Degradation of     gallic acid and hydrolysable polyphenols is constitutively activated     in the freshwater plant-associated bacterium Matsuebacter sp FB25.     Aquat Microb Ecol 47, 83-90. -   23. Yun C S, Amakata D, Matsuo Y, Matsuda H & Kawamukai M (2005) New     chitosan-degrading strains that produce chitosanases similar to ChoA     of Mitsuaria chitosanitabida. Appl Environ Microbiol 71, 5138-5144. -   24. Kaiser O, Puhler A & Selbitschka W (2001) Phylogenetic analysis     of microbial diversity in the rhizoplane of oilseed rape (Brassica     napus cv. westar) employing cultivation-dependent and     cultivation-independent approaches. Microb Ecol 42, 136-149. -   25. Gomila M, et al (2005) Identification of culturable bacteria     present in haemodialysis water and fluid. FEMS Microbiol Ecol 52,     101-114. -   26. Malmqvist A, et al (1994) Ideonella dechloratans gen. nov., sp.     nov., a new bacterium capable of growing anaerobically with chlorate     as an electron acceptor. Syst Appl Microbiol 17, 58. -   27. Siering P L & Ghiorse W C (1996) Phylogeny of the     Sphaerotilus-Leptothrix group inferred from morphological     comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence     analyses. Int J Syst Bacteriol 46, 173-182. -   28. Xie C H & Yokota A (2005) Reclassification of Alcaligenes latus     strains IAM 12599 (T) and IAM 12664 and Pseudomonas saccharophila as     Azohydromonas lata gen. nov., comb. nov., Azohydromonas australica     sp nov and Pelomonas saccharophila gen. nov., comb. nov.,     respectively. Int J Syst Evol Microbiol 55, 2419-2425. -   29. Gomila M, Bowien B, Falsen E, Moore E R B & Lalucat J (2008)     Description of Roseateles aquatilis sp. nov. and Roseateles terrae     sp. nov., in the class Betaproteobacteria, and emended description     of the genus Roseateles. Int J Syst Evol Microbiol 58, 6-11. -   30. Kadivar H & Stapleton A (2003) Ultraviolet radiation alters     maize phyllosphere bacterial diversity. Microb Ecol 45, 353. -   31. Roesch L, Camargo F, Bento F & Triplett E (2008) Biodiversity of     diazotrophic bacteria within the soil, root and stem of field-grown     maize. Plant Soil 302, 91-104. -   32. Coelho R, et al (2008) Diversity of nifH gene pools in the     rhizosphere of two cultivars of sorghum (Sorghum bicolor) treated     with contrasting levels of nitrogen fertilizer. FEMS Microbiol Lett     279, 15. -   33. Macur R E, Wheeler J T, Burr M D & Inskeep W P (2007) Impacts of     2,4-D application on soil microbial community structure and on     populations associated with 2,4-D degradation. Microbiological     Research, 162, 37-45. -   34. Hayatsu M, Hirano M & Tokuda S (2000) Involvement of two     plasmids in fenitrothion degradation by Burkholderia sp. strain     NF100. Appl Environ Microbiol 66, 1737-1740. -   35. Kikuchi Y, Hosokawa T & Fukatsu T (2007) Insect-microbe     mutualism without vertical transmission: A stinkbug acquires a     beneficial gut symbiont from the environment every generation. Appl     Environ Microbiol 73, 4308-4316. -   36. Murray R & Stackebrandt E (1995) Taxonomic note: Implementation     of the provisional status Candidatus for incompletely described     procaryotes. Int J Syst Bacteriol 45, 186-187. -   37. Coenye T & Vandamme P (2003) Diversity and significance of     Burkholderia species occupying diverse ecological niches. Environ     Microbiol 5, 719-729. -   38. Parke J L & Gurian-Sherman D (2001) Diversity of the     Burkholderia cepacia complex and implications for risk assessment of     biological control strains. Annu Rev Phytopathol 39, 225-258. -   39. el-Banna N & Winkelmann G (1998) PyrroInitrin from Burkholderia     cepacia: Antibiotic activity against fungi and novel activities     against streptomycetes. J Appl Microbiol 85, 69. -   40. Caballero-Mellado J, Onofre-Lemus J, Estrada-de los Santos P &     Martinez-Aguilar L (2007) The tomato rhizosphere, an environment     rich in nitrogen-fixing Burkholderia species with capabilities of     interest for agriculture and bioremediation. Appl Environ Microbiol     73, 5308-5319. -   41. Perin L, et al (2006) Diazotrophic Burkholderia species     associated with field-grown maize and sugarcane. Appl Environ     Microbiol 72, 3103-3110. -   42. Salles J F, Samyn E, Vandamme P, van Veen J A & van Elsas J     D (2006) Changes in agricultural management drive the diversity of     Burkholderia species isolated from soil on PLAT medium. Soil Biology     & Biochemistry 38, 661-673. -   43. Salles J F, van Elsas J D & van Veen J A (2006) Effect of     agricultural management regime on Burkholderia community structure     in soil. Microb Ecol 52, 267-279. -   44. Bankhead S B, Landa B B, Lutton E, Weller D M & McSpadden     Gardener B B (2004) Minimal changes in rhizobacterial population     structure following root colonization by wild type and transgenic     biocontrol strains. FEMS Microbiol Ecol 49, 307-318. -   45. McSpadden Gardener B B & Weller D M (2001) Changes in     populations of rhizosphere bacteria associated with take-all disease     of wheat. Appl Environ Microbiol 67, 4414-4425. -   46. Nakanishi Y, et al (2006) Increase in terminal restriction     fragments of bacteroidetes-derived 16S rRNA genes after     administration of short-chain fructooligosaccharides. Appl Environ     Microbiol 72, 6271-6276. -   47. Widmer F, Hartmann M, Frey B & Kolliker R (2006) A novel     strategy to extract specific phylogenetic sequence information from     community T-RFLP. J Microbiol Methods 66, 512-520. -   48. Cadillo-Quiroz H, Yashiro E, Yavitt J B & Zinder S H (2008)     Characterization of the archaeal community in a minerotrophic fen     and Terminal restriction fragment length polymorphism-directed     isolation of a novel hydrogenotrophic methanogen. Appl Environ     Microbiol 74, 2059-2068. -   49. Jeon C O, et al (2003) Discovery of a bacterium, with     distinctive dioxygenase, that is responsible for in situ     biodegradation in contaminated sediment. Proceedings of the National     Academy of Sciences 100, 13591-13596. -   50. Yin B, Valinsky L, Gao X B, Becker J O & Borneman J (2003)     Identification of fungal rDNA associated with soil suppressiveness     against Heterodera schachtii using oligonucleotide fingerprinting.     Phytopathology 93, 1006-1013. -   51. Olatinwo R, Yin B, Becker J O & Borneman J (2006) Suppression of     the plant-parasitic nematode Heterodera schachtii by the fungus     Dactylella oviparasitica. Phytopathology 96, 111-114. -   52. Valinsky, et al (2002) Analysis of bacterial community     composition by oligonucleotide fingerprinting of rRNA genes. Appl     Environ Microbiol 68, 3243. -   53. Leveau J H J (2007) The magic and menace of metagenomics:     Prospects for the study of plant growth-promoting rhizobacteria. Eur     J Plant Pathol 119, 279-300. -   54. Atlas, R M (1997) Handbook of Microbiological Media, ed     Lawrence C. Parks (CRC Press Inc., United States of America), pp     1706. -   55. Kampfer P, et al (1996) Characterization of bacterial     communities from activated sludge: Culture-dependent numerical     identification versus in situ identification using group- and     genus-specific rRNA-targeted oligonucleotide probes. Microb Ecol 32,     101-121. -   56. Massa S, Caruso M, Trovatelli F & Tosques M (1998) Comparison of     plate count agar and R2A medium for enumeration of heterotrophic     bacteria in natural mineral water. World J Microbiol Biotechnol 14,     727-730. -   57. Kampfer P (1997) Detection and cultivation of filamentous     bacteria from activated sludge. FEMS Microbiol Ecol 23, 169-181. -   58. Bergey's manual of systematic bacteriology (2005), eds Boone D     R, Castenholz R W & Garrity G M (Springer, New York). -   59. Chapin L, Wang Y, Lutton E, McSpadden Gardener B B (2006)     Distribution and fungicide sensitivity of fungal pathogens causing     anthracnose-like lesions on tomatoes grown in Ohio. Plant Dis 90,     397.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

1. A composition comprising about 10³ cfu to about 10¹¹ cfu of a bacterial strain per gram dry inert carrier, wherein said bacterial strain is designated from the group consisting of H24L5A, deposited as ATCC Accession No. PTA-10183, and R4F2, deposited as ATCC Accession No. PTA-10182.
 2. The composition of claim 1, wherein said bacterial strain is designated R4F2, deposited as ATCC Accession No. PTA-10182.
 3. The composition of claim 1, further comprising about 1% to about 40% growth medium per gram of the carrier on a wt/wt dry basis.
 4. The composition of claim 1, wherein said bacterial strain exhibits plant pathogen suppression.
 5. The composition of claim 1, wherein said bacterial strain exhibits fungicidal or fungistatic activity when applied to plant material or the soil environment.
 6. The composition of claim 1, wherein said bacterial strain exhibits fungicidal or fungistatic activity towards a fungal or oomycete plant pathogen in situ. 7-11. (canceled)
 12. A method of controlling the growth of a plant pathogenic fungus, comprising applying to a plant a composition comprising a bacterial strain, the composition exhibits fungicidal or fungistatic activity towards said plant pathogenic fungus, wherein symptoms of a disease caused by said fungus are suppressed on said plant, and wherein the bacterial strain is selected from a group consisting of the bacterial strain designated H24L5A, deposited as ATCC Accession No. PTA-10183; the bacterial strain designated R4F2, deposited as ATCC Accession No. PTA-10182; the bacterial strain comprises a nucleic acid, the nucleic acid comprising a sequence at least 97% identical to SEQ ID NO: 10; the bacterial strain comprises a nucleic acid, the nucleic acid comprising a sequence at least 97% identical to SEQ ID NO: 19; the bacterial strain comprises genomic DNA with a 16S sequence indicative of the bacterial species of the strain designated H24L5A, deposited as ATCC Accession No. PTA-10183, and the bacterial strain comprises genomic DNA with a 16S sequence indicative of the bacterial species of the strain designated R4F2, deposited as ATCC Accession No. PTA-10182.
 13. (canceled)
 14. The composition of claim 2, further comprising about 1% to about 40% growth medium per gram of the carrier on a wt/wt dry basis.
 15. The composition of claim 2, wherein said bacterial strain exhibits plant pathogen suppression.
 16. The composition of claim 2, wherein said bacterial strain exhibits fungicidal or fungistatic activity when applied to plant material or the soil environment.
 17. The composition of claim 2, wherein said bacterial strain exhibits fungicidal or fungistatic activity towards a fungal or oomycete plant pathogen in situ. 